Variations in Essential Oils from the Leaves of Cinnamomum bodinieri in China

Cinnamomum plants are rich in natural essential oils, which are widely used as materials in the fragrance, insecticidal, antibacterial agent, pharmaceutical, and food industries; however, few studies have investigated the essential oil components of Cinnamomum bodinieri. Therefore, this study investigated the diversity of essential oils from the leaves of 885 individual C. bodinieri plants across 32 populations in five provinces. Essential oils were extracted by hydrodistillation, and then qualitative and quantitative analyses of the compounds were performed by GC-MS and GC-FID. A total of 87 chemical constituents were identified in the essential oils, including 33 monoterpenes, 48 sesquiterpenes, and six other compounds. The average oil yield was 0.75%, and individual oil yields ranged from 0.01% to 4.28%. A total of 16 chemotypes were classified according to variations in the essential oil chemical constituents of C. bodinieri, among which the camphor-type, citral-type, and eucalyptol-type were dominant. Moreover, the borneol-type, cymol-type, elemol-type, methylisoeugenol-type, and selina-6-en-4-ol-type were reported in C. bodinieri for the first time. The yield and principal components of the essential oils were mainly affected by altitude, temperature, and sunshine duration, among which altitude had the most significant effect; thus, low-altitude areas are more suitable for the synthesis and accumulation of essential oils. Based on the different characteristics of the essential oils in the leaves of C. bodinieri, several excellent populations and individuals were identified in this study. Moreover, the findings provide a foundation for breeding superior varieties and studying essential oil biosynthesis mechanisms in the future.


Introduction
Cinnamomum bodinieri is an evergreen broad-leaved tree of the Lauraceae that is distributed only in limestone or karst areas in the western Hunan, Guizhou, Hubei, Sichuan, and eastern Yunnan provinces in China [1]. The branches and leaves of Cinnamomum plants are rich in natural essential oils, which can be widely used as materials in the fragrance, insecticidal, antibacterial agent, pharmaceutical, and food industries [2][3][4]. At present, Cinnamomum camphora is widely used in the essential oil industry and has been investigated to determine whether Cinnamomum plants can be cultivated in forests as a raw material for the production of certain spices; however, the development and application of essential oils from other Cinnamomum plants are relatively limited [5]. Compared with C. camphora, C. bodinieri has the advantages of larger biomass, stronger stress resistance, and more rapid growth, which are important for the development and utilisation of natural Cinnamomum essential oils.
Current research on C. bodinieri is mainly focused on the stress resistance of the plant and breeding technology [6][7][8][9]. However, few studies have reported on the essential oils of C. bodinieri. Relevant studies have extracted and analysed the essential oils from the leaves of C. bodinieri and found that they are mainly composed of citral and have strong antioxidant biological activity [10]. The essential oils of plants have complex and diverse compositions, and their synthesis, accumulation, and transformation are affected by genetic and environmental factors [11,12]. Moreover, the chemical composition of essential oils from different individuals of the same species may vary greatly; for example, the essential oil of C. camphora has a variety of chemotypes, such as camphor, borneol, and linalool [13,14].
However, the leaf essential oils of only a small number of individuals of C. bodinieri have been analysed, and the samples were not collected over a large area. Moreover, relevant reports on the diversity of essential oil from the leaves of C. bodinieri are not available, and many new chemotypes of the essential oils of C. bodinieri remain to be discovered. Therefore, this study investigated 32 natural populations of C. bodinieri in its main distribution areas in China, such as Hunan, Hubei, Guizhou, Sichuan, and Yunnan. In total, 885 C. bodinieri leaf samples were collected. The yield, chemical composition, and chemotypes of the essential oils and influential environmental factors were analysed to explore the diversity of the oils and determine the law of population geographic variations. The results provide an important theoretical basis for the breeding, development, and utilisation of C. bodinieri essential oils.

Variation in the Essential Oil Yield
Essential oils from 885 C. bodinieri individuals from 32 populations were extracted and analysed. A frequency distribution map was created using 0.2% as the distribution interval ( Figure 1). The results showed that the individual C. bodinieri decreased gradually with the increase in the oil yield interval, and the individual C. bodinieri oil yield interval was mainly concentrated in the two intervals below 0.4. The largest number of individuals of C. bodinieri was 233 in the 0-0.2 interval, followed by 194 in the 0.2-0.4 interval. A total of 427 were found in the first two intervals, which represented 48.2% of the total number. Compared with the previous two intervals, the number of individuals in the 0.4-2.0 interval decreased significantly and ranged from 20 to 90, and the number of individuals in the 2.0-3.0 interval was 5 to 20. The oil yield of a few individuals could exceed 3.0. The highest oil yield could reach 4.28%, while the lowest oil yield was only 0.01%, which represented a difference of 428 times. The great differences between individuals indicate that C. bodinieri leaves are naturally extremely rich in essential oil genetic resources, thus indicating that this plant has great potential for breeding.

Composition and Correlations of the Main Chemical Constituents
A total of 87 compounds were identified by statistical analysis of the leaf components of wild C. bodinieri populations, and they included 12 hydrocarbon monoterpenes, 21 oxygen-containing monoterpenes, 23 hydrocarbon sesquiterpenes, 25 oxygen-containing sesquiterpenes, and six other compounds ( Figure 3a). analysed ( Figure 3b). The results showed that neral and geranial in C. bodinieri essential oil were significantly correlated and often found together in the essential oils. Β-Phellandrene, eucalyptol, and 4-terpineol were significantly positively correlated; carvone, trans-nerolidol, and β-carophyllene were significantly positively correlated; and spathulenol and carophylene oxide were significantly positively correlated. Camphor was negatively correlated with eucalyptol, linalool, neral, geranial, and trans-nerolidol, and linalool was negatively correlated with eucalyptol, neral, and geranial.

Chemotype Classification and Principal Component Analysis
The chemical composition of the essential oils from 885 C. bodinieri plants from 32 populations was statistically analysed based on a principal component analysis ( Figure  4). The first four principal components (PC1, PC2, PC3, and PC4) accounted for 45.3%, 18.6%, 11.6%, and 9.6% of the variation, respectively, which represented 85.1% of the total variation. Camphor was clearly separated from linalool based on PC1 and PC2; camphor was positively correlated with PC1 and negatively correlated with PC2, and linalool was negatively correlated with PC1 and PC2. PC3 and PC4 could separate eucalyptol, trans-nerolidol, and citral, in which eucalyptol was positively correlated with PC3 and PC4, trans-nerolidol was negatively correlated with PC3 and PC4, and citral was negatively correlated with PC3 and positively correlated with PC4. The principal component analysis revealed that 885 individual C. bodinieri plants could be divided into seven types, namely, camphor-type, citral-type, eucalyptol-type, linalool-type, trans-nerolidol-type, mixed-type (principal component content < 30%), and other chemotypes. Correlations between the 20 chemical components with the highest frequencies were analysed ( Figure 3b). The results showed that neral and geranial in C. bodinieri essential oil were significantly correlated and often found together in the essential oils. B-Phellandrene, eucalyptol, and 4-terpineol were significantly positively correlated; carvone, trans-nerolidol, and β-carophyllene were significantly positively correlated; and spathulenol and carophylene oxide were significantly positively correlated. Camphor was negatively correlated with eucalyptol, linalool, neral, geranial, and trans-nerolidol, and linalool was negatively correlated with eucalyptol, neral, and geranial.

Chemotype Classification and Principal Component Analysis
The chemical composition of the essential oils from 885 C. bodinieri plants from 32 populations was statistically analysed based on a principal component analysis ( Figure 4). The first four principal components (PC1, PC2, PC3, and PC4) accounted for 45.3%, 18.6%, 11.6%, and 9.6% of the variation, respectively, which represented 85.1% of the total variation. Camphor was clearly separated from linalool based on PC1 and PC2; camphor was positively correlated with PC1 and negatively correlated with PC2, and linalool was negatively correlated with PC1 and PC2. PC3 and PC4 could separate eucalyptol, transnerolidol, and citral, in which eucalyptol was positively correlated with PC3 and PC4, trans-nerolidol was negatively correlated with PC3 and PC4, and citral was negatively correlated with PC3 and positively correlated with PC4. The principal component analysis revealed that 885 individual C. bodinieri plants could be divided into seven types, namely, camphor-type, citral-type, eucalyptol-type, linalool-type, trans-nerolidol-type, mixed-type (principal component content <30%), and other chemotypes.
A significant difference was observed in the oil yields of some chemotypes of C. bodinieri (p < 0.05). The camphor-type had the highest oil yield, with an average of 1.18%, followed by linalool-type, eucalyptol-type, and selina-6-en-4-ol-type, with averages of 1.06%, 0.73%, 0.95%, and 0.73%, respectively. The safrole-type oil yield was the lowest (0.08%). The oil yields and relative principal component values of different individuals of the same chemotype were significantly different. For example, the oil yield of the camphor-type ranged from 0.02% to 4.28%, and the relative principal component value was 30.42-98.52%; the oil yield of the linalool-type ranged from 0.01 to 2.73%, and the relative principal component value was 31.39-97.14%; and the oil yield of the citral-type ranged from 0.02 to 1.51%, and the relative principal component value was 31.77-89.12%.
Although the chemotypes of the essential oil of C. bodinieri were mainly camphor-type, linalool-type, citral-type, eucalyptol-type, trans-nerolidol-type, and mixed-type, other chemotypes were observed. These chemotypes were very rare and found in less than 20 plants, while some chemotypes were only found in one plant. However, most of these chemotypes were unique chemical types discovered for the first time in C. bodinieri, especially in certain individuals with high relative principal compo-    A significant difference was observed in the oil yields of some chemotypes of C. bodinieri (p < 0.05). The camphor-type had the highest oil yield, with an average of 1.18%, followed by linalool-type, eucalyptol-type, and selina-6-en-4-ol-type, with averages of 1.06%, 0.73%, 0.95%, and 0.73%, respectively. The safrole-type oil yield was the lowest (0.08%). The oil yields and relative principal component values of different individuals of the same chemotype were significantly different. For example, the oil yield of the camphor-type ranged from 0.02% to 4.28%, and the relative principal component value was 30.42-98.52%; the oil yield of the linalool-type ranged from 0.01 to 2.73%, and the relative principal component value was 31.39-97.14%; and the oil yield of the citral-type ranged from 0.02 to 1.51%, and the relative principal component value was 31.77-89.12%.
Although the chemotypes of the essential oil of C. bodinieri were mainly camphortype, linalool-type, citral-type, eucalyptol-type, trans-nerolidol-type, and mixed-type, other chemotypes were observed. These chemotypes were very rare and found in less than 20 plants, while some chemotypes were only found in one plant. However, most of these chemotypes were unique chemical types discovered for the first time in C. bodinieri, especially in certain individuals with high relative principal component loadings, including The proportion of different chemotypes in each C. bodinieri population was analysed ( Figure 5). The results showed that the camphor-type existed in all investigated populations and was dominant in GZ-WA, GZ-XX, HN-GZ, HN-HY, GZ-LS, and GZ-LD. Therefore, the camphor-type was the most abundant and widely distributed chemotype in the C. bodinieri populations. Second, the main chemotype of GZ-DY and GZ-GSH was the linalool-type, which accounted for more than 90%, although a few individual plants reached 97%. In addition, the oil yield of these populations reached 2.1%. The GZ-NM, GZ-ZJ, and GZ-ZY populations were dominated by the citral-type, GZ-TR by the trans-nerolidol-type, and GZ-MJ was dominated by the mixed-type. In addition, the chemotypes of the GZ-WA, GZ-DY, and HN-HY populations were relatively small, with only 1-3 chemotypes in the populations, while chemotypes of HB-BK, HB-JS, GZ-ZY, GZ-XE, HN-JSS, and GZ-PD were more abundant The proportion of different chemotypes in each C. bodinieri population was analysed ( Figure 5). The results showed that the camphor-type existed in all investigated populations and was dominant in GZ-WA, GZ-XX, HN-GZ, HN-HY, GZ-LS, and GZ-LD. Therefore, the camphor-type was the most abundant and widely distributed chemotype in the C. bodinieri populations. Second, the main chemotype of GZ-DY and GZ-GSH was the linalool-type, which accounted for more than 90%, although a few individual plants reached 97%. In addition, the oil yield of these populations reached 2.1%. The GZ-NM, GZ-ZJ, and GZ-ZY populations were dominated by the citral-type, GZ-TR by the trans-nerolidol-type, and GZ-MJ was dominated by the mixed-type. In addition, the chemotypes of the GZ-WA, GZ-DY, and HN-HY populations were relatively small, with only 1-3 chemotypes in the populations, while chemotypes of HB-BK, HB-JS, GZ-ZY, GZ-XE, HN-JSS, and GZ-PD were more abundant

Correlation between C. bodinieri Essential Oils and Environmental Factors
The correlation between the oil yield of C. bodinieri leaves and the relative contents of the seven main chemotypes and geographical environmental factors was analysed ( Table  2). The results showed that the oil yield was significantly negatively correlated with altitude and positively correlated with annual rainfall and average temperature. The rela-

Correlation between C. bodinieri Essential Oils and Environmental Factors
The correlation between the oil yield of C. bodinieri leaves and the relative contents of the seven main chemotypes and geographical environmental factors was analysed ( Table 2). The results showed that the oil yield was significantly negatively correlated with altitude and positively correlated with annual rainfall and average temperature. The relative camphor, eucalyptol, linalool, and citral contents were significantly negatively correlated with altitude. The relative citral content was positively correlated with the annual average temperature. The relative elemol, cymol, and camphor contents were positively correlated with the annual average sunshine length. Comprehensive analysis showed that latitude and longitude were not the main factors affecting the essential oil of C. bodinieri, although altitude, temperature, and sunshine length had significant effects and altitude difference had the most obvious effect. The yield of C. bodinieri essential oil and relative content of the main components, such as camphor-type, citral-type, and linalool-type, were significantly higher in low-altitude areas than high-altitude areas.

Discussion
Cinnamomum plants usually contain natural essential oils in their roots, stems, and leaves, and some of these species have been widely cultivated as important spice trees, thus forming a complete industrial chain that integrates planting, harvesting, extraction, and processing [15,16]. The essential oil of Cinnamomum plants has abundant chemical components and thus has been studied by many domestic and foreign scholars. Researchers have studied the diversity of essential oils from Cinnamomum parthenoxylon leaves and classified the oils into 14 chemotypes. The dominant chemotypes are the camphor-type and linalool-type [17]. The main components in the essential oil of Cinnamomum zeylanicum leaves are E-cinnamaldehyde and eugenol [18]; the main component in the essential oil of Cinnamomum longipaniculatum is eucalyptol [19]; and the main component of the essential oil of Cinnamomum pauciflorum leaves is safrole [20].
In this study on the diversity of leaf essential oils from 32 populations of C. bodinieri, essential oils were divided into 16 chemotypes. The essential oil chemotype contents of C. bodinieri are among the most abundant of Cinnamomum plants, and the dominant chemotypes are camphor-type, citral-type, and eucalyptol-type. Camphor is a common chemical component of Cinnamomum plants [21], the camphor-type was found in the largest number of individuals among the C. bodinieri populations. The individual oil yield varied greatly, with the highest oil yield of 4.28%, which was significantly higher than that reported for C. camphora (3.46%) [22]. Citral essential oil is mainly found in the fruit of Litsea cubeba and rarely occurs as the main component in the essential oils of C. camphora, C. longipaniculatum, C. parthenoxylon, and other Cinnamomum plants [23,24]; however, 160 citral-type individuals were found in the currently studied C. bodinieri population, thus accounting for 16.7% of the total number of individuals, which was significantly higher than that of other Cinnamomum species. Eucalyptol mainly exists in eucalyptus plants and has many functions, such as anti-inflammatory, analgesic, antibacterial, and tumour cell inhibition; thus, it is primarily used in industrial raw materials and medicine [25,26]. The relative content of eucalyptus in the essential oil of C. bodinieri was as high as 67.09%, and the highest oil yield was 2.86%, which was higher than that of most eucalyptus plants. Therefore, these three excellent chemotypes should be screened in C. bodinieri to determine the potential use of this species in forests to generate spice raw material. In addition, studies on C. camphora, C. parthenoxylon, Cinnamomum tenuipilum, and Cinnamomum pauciflorum have reported the dominance of the borneol-type, cymol-type, elemol-type, and methylisoeugenol-type [27]; however, this study provides the first report of these chemotypes in C. bodinieri.
The essential oils of Cinnamomum are primarily monoterpenes, sesquiterpenes, and phenylpropanoids. Monoterpene biosynthesis in plants mainly originates from the methylerythritol phosphate pathway (MEP), sesquiterpene biosynthesis mainly originates from the mevalonate pathway (MVA), and phenylpropanoid synthesis mainly originates from the shikimate pathway [28]. A total of 87 chemical constituents were detected in the essential oil of C. bodinieri leaves, including 33 monoterpenes, 48 sesquiterpenes, and six other compounds. These chemical components are related to each other, indicating the complexity of the formation mechanism of the essential oils of Cinnamomum. An indepth study of the genome and transcriptome of Lauraceae species, such as C. camphora, L. cubeba, and C. parthenoxylon, has made important breakthroughs in the study of terpenoid biosynthesis pathways and mechanisms, such as linalool, camphor, eucalyptol, transnerolidol, and citral [29][30][31]. Few studies on Cinnamomum have investigated terpenoids, such as α-phellandrene and cymol, and phenylpropanoid compounds, such as safrole and methylisoeugenol; therefore, the discovery of new chemotypes in C. bodinieri provides an important basis for further studies on the biosynthesis mechanism of essential oils in Cinnamomum.
Significant differences were observed in the essential oils of C. bodinieri leaves among the different populations. Most populations were dominated by the camphor-type; GZ-DY and GZ-GSH were dominated by the linalool-type; GZ-NM, GZ-ZJ, and GZ-ZY were dominated by the citral-type; and YN-WX was dominated by the eucalyptol-type. The HB-BK, HB-JS, GZ-ZY, GZ-XE, HN-JSS, and GZ-PD communities are abundant and can be used as candidate populations for breeding new chemotypes. Moreover, the difference in essential oils between individuals in the C. bodinieri populations are also very significant, and this phenomenon of significant differences within and between populations has also been noted in studies on the diversity of leaf essential oils of other Cinnamomum plants [32,33]. Therefore, the diversity between populations and individuals should be fully considered when selecting essential oil resources from Cinnamomum.
The synthesis and accumulation of plant essential oils are affected by multiple genetic and environmental factors, including altitude, longitude, latitude, temperature, and rainfall [34,35]. The essential oil yields of Thymus carmannicus and Oliveria decumbens gradually decreased with increasing altitude [36,37]. The accumulation of Lavandula latifolia essential oil was mainly affected by latitude and rainfall [38]. The correlation analysis between the essential oils of C. bodinieri leaves and environmental factors showed that altitude, sunshine duration, and annual average temperature had the greatest effects, while longitude and latitude had weaker effects. The climate at low altitudes in southern China is warm and humid, which can significantly increase the yield of volatile oil from C. bodinieri leaves. Although the natural distribution of C. bodinieri is mainly in the southwest of China, according to its adaptability to environmental factors, it can be introduced to the southern and southeastern low-altitude areas of China, such as Jiangxi, Fujian, and Guangdong, to improve the yield of C. bodinieri essential oil.
Samples were collected by random sampling, and 200-300 g of leaf tissue was collected from individual plants. The tissue samples were then mixed, weighed evenly, and stored in an airtight container and used to record and collect geographic location information (Table 3, Figure 6). All collected plant materials were identified by Dr. Xie Yifei of Gannan Normal University and stored in the Nanling Herbarium.

Distillation of Essential Oil
The essential oils were extracted and collected from each of the samples by reflux hydrodistillation. The leaf samples (200-300 g) were placed in the device (Reflux steam distillation apparatus) and purified water (sample weight/water weight = 1:10) was added. After 2 h of steam distillation, the essential oils were obtained by the liquid separator on the device. Water in the essential oils was removed with anhydrous sodium sulphate, and the weights of the essential oils were measured using an electronic balance. The extracted essential oils were dissolved in anhydrous ethanol (essential oil/ethanol = 30:970) and analysed using GC-MS and GC-FID.

Essential Oil Chemical Composition Analysis
QP2020 GC-MS (Shimadzu, Kyoto, Japan) was used in this test. ASH-RXI-5SILMS column (30 m × 0.25 mm × 0.25 μm) was used as carrier gas with ultra-pure helium gas. Flow rate 1.0 mL/min, injection port temperature 200 °C, injection volume 1.0 μL, dplit ratio 20:1, EI ion source temperature 280 °C, scanning mass range 50-650 m/z. The initial temperature of GC was 60 °C for 2 min, and the GC was heated up at 5 °C/min to 220 °C and maintained for 20 min. The GC-MS data processing system was used to search the

Distillation of Essential Oil
The essential oils were extracted and collected from each of the samples by reflux hydrodistillation. The leaf samples (200-300 g) were placed in the device (Reflux steam distillation apparatus) and purified water (sample weight/water weight = 1:10) was added. After 2 h of steam distillation, the essential oils were obtained by the liquid separator on the device. Water in the essential oils was removed with anhydrous sodium sulphate, and the weights of the essential oils were measured using an electronic balance. The extracted essential oils were dissolved in anhydrous ethanol (essential oil/ethanol = 30:970) and analysed using GC-MS and GC-FID.

Essential Oil Chemical Composition Analysis
QP2020 GC-MS (Shimadzu, Kyoto, Japan) was used in this test. ASH-RXI-5SILMS column (30 m × 0.25 mm × 0.25 µm) was used as carrier gas with ultra-pure helium gas.
Flow rate 1.0 mL/min, injection port temperature 200 • C, injection volume 1.0 µL, dplit ratio 20:1, EI ion source temperature 280 • C, scanning mass range 50-650 m/z. The initial temperature of GC was 60 • C for 2 min, and the GC was heated up at 5 • C/min to 220 • C and maintained for 20 min. The GC-MS data processing system was used to search the database (NIST 8.0), and a series of n-alkanes (C8-32, Sigma-Aldrich, St. Louis, MO, USA) retention indices under the same conditions were compared with the relevant literature to identify the compounds [39].
Quantitative analyses were performed using the GC-FID method on a GC-2010 Plus (Shimadzu, Kyoto, Japan) equipped with the same column as GC-MS. The conditions were the same as GC-MS except that N2 gas was used as carrier gas. GC data processing system was used to calculate the relative content of compounds. Relative content (%) = 100 × compound peak area/total peak area.

Data Analysis
The natural population distribution map of C. bodinieri was drawn using ArcGIS (Version 10.0; Esri, Redlands, CA, USA), the oil yield frequency was statistically analysed by Origin (Version 19.0; OriginLab, Northampton, MA, USA), and the chemical population distribution, principal component analysis (PCA), and chemical component correlation heat map were mapped using the ggplot and pca package analysis tools in R language. The correlation analysis of essential oil characteristics with environmental and climatic factors was performed using SPSS software (version 22.0; SPSS Inc, Chicago, IL, USA)

Conclusions
Sixteen chemotypes were observed in the essential oil from Chinese C. bodinieri leaves, among which the camphor-type, citral-type, and eucalyptol-type were dominant and found in the largest number of individuals. Certain chemotypes, such as the trans-nerolidol-type, borneol-type, cymol-type, elemol-type, methyl isoeugenol-type, and selina-6-en-4-ol-type, were first reported in C. bodinieri, and these results can provide a basis for studying the biosynthesis mechanism of Cinnamomum essential oil and breeding improved varieties. The average oil yield of C. bodinieri leaves was 0.75%, and the oil yield among individuals showed a difference of more than 400 times. The oil yields of the GZ-WA, GZ-DY, and GZ-LS communities were the highest, with an average yield of more than 1.5%. In addition, the yield and main component contents of C. bodinieri leaf essential oil were mainly affected by altitude, sunshine duration, and temperature. Low-altitude and warm climate areas are more suitable for the synthesis of essential oil in this species.